Method of stabilizing oxidation color former

ABSTRACT

A method of stabilizing an oxidation color former in a solution is provided. N-(carboxymethylaminocarbonyl)-4,4′-bis(dimethylamino) diphenylamine sodium salt as an oxidation color former is stabilized by providing it with at least one of a fructosyl amino acid oxidase (FAOD) and a peroxidase (POD) in the solution. The concentration of the FAOD is in the range from 0.01 to 1.0 g/l or 1 to 100 KU/l, and the concentration of the POD is in the range from 0.01 to 1.0 g/l or 1 to 100 KU/l.

TECHNICAL FIELD

[0001] The present invention relates to a method of stabilizing anoxidation color former in a solution and an oxidation color formerreagent using the method.

BACKGROUND ART

[0002] The use of N-(carboxymethylaminocarbonyl)-4,4′-bis(dimethylamino)diphenylamine sodium salt as a highly sensitive color former thatdevelops color by oxidation has been known generally. Such a highlysensitive color former can be used, for example, when determining theamount of an oxidizing substance by causing a reaction between the colorformer and the oxidizing substance using an oxidoreductase and thenmeasuring an amount of the color developed by measuring an absorbance.When N-(carboxymethylaminocarbonyl)-4,4′-bis(dimethylamino)diphenylamine sodium salt is to beused in such a redox reaction, a solution prepared by dissolving it inwater usually is used as a liquid reagent.

[0003] However, an oxidation color former such as N-(carboxymethylaminocarbonyl)-4,4′-bis(dimethylamino)diphenylamine sodium salt is unstablein an aqueous solution and thus may develop color spontaneously within aday after the preparation of the aqueous solution. Therefore, whenN-(carboxy methylaminocarbonyl)-4,4′-bis (dimethylamino) diphenylaminesodium salt stored as a solution is used, there arises a problem in thata background absorbance increases in the measurement of an absorbance,thereby degrading the accuracy of the measurement.

[0004] In order to prevent the influence of such spontaneous colordevelopment, it is necessary to prepare a liquid reagent for eachmeasurement. However, this makes the operation complicated and resultsin high cost.

DISCLOSURE OF INVENTION

[0005] Therefore, it is an object of the present invention to provide amethod of stabilizing an oxidation color former such asN-(carboxymethylamino carbonyl)-4,4′-bis(dimethylamino)diphenylaminesodium salt in a solution and a reagent using the method.

[0006] In order to achieve the above object, the present inventionprovides a method of stabilizing an oxidation color former in asolution, including: making at least one of a fructosyl amino acidoxidase (hereinafter referred to as “FAOD”) and a peroxidase(hereinafter referred to as “POD”) present with the oxidation colorformer in the solution. The oxidation color former may be, for example,N-(carboxymethylaminocarbonyl)-4,4′-bis(dimethylamino) diphenylaminesodium salt.

[0007] When at least one of the FAOD and the POD is present with theoxidation color former in the solution, spontaneous color development ofthe oxidation color former such asN-(carboxymethylaminocarbonyl)-4,4′-bis(dimethylamino)diphenylaminesodium salt can be suppressed, although the mechanism is unknown. Thisallows the oxidation color former to be stored as a solution, forexample. Therefore, the necessity of preparing a liquid reagent for eachmeasurement is eliminated, which allows measurement or the like to becarried out simply and at low cost. Moreover, when the stored solutionis used as a reagent causing a color-developing reaction, an increase inbackground absorbance in the measurement of an absorbance is suppressedso that the accuracy of the measurement can be improved.

[0008] In the method of the present invention, it is preferable that theconcentration of the oxidation color former is in a range from 1 to10,000 μmol/l.

[0009] In the method of the present invention, the concentration of theFAOD preferably is in a range from 0.002 to 200 g/l or 0.1 to 1000 KU/l,and the concentration of the POD preferably is in a range from 0.02 to50 g/l or 1 to 5000 KU/l.

[0010] In the method of the present invention, the amount of the FAODpreferably is in a range from 0.01 to 200 g or 0.5 to 1000 KU per 0.1mmol of the oxidation color former, and the amount of the POD preferablyis in a range from 0.02 to 50 g or 1 to 5000 KU per 0.1 mmol of theoxidation color former. When both the enzymes are added, preferably theyare added so that the amount of the FAOD is in a range from 0.01 to 100g or 0.5 to 600 KU while the amount of the POD is in a range from 0.02to 50 g or 1 to 5000 KU.

[0011] In the method of the present invention, it is preferable that thesolution contains at least one buffer selected from the group consistingof ADA buffer, Tris-HCl buffer, Bis-Tris buffer, glycylglycine buffer,Bicine buffer, and phosphate buffer because this allows the spontaneouscolor development to be suppressed more strongly.

[0012] In the method of the present invention, it is preferable that atleast one antioxidant selected from the group consisting of α-tocopherolacetate (VE), potassium erythorbate, and potassium sorbate; at least onechelating agent selected from the group consisting ofethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaaceticacid (DTPA), trans-1,2-diaminocyclohexane-N, N, N′,N′-tetraacetic acid(CyDTA), O,O′-bis (2-aminoethyl)ethyleneglycol-N,N,N′,N′-tetraaceticacid (GEDTA), and nitrilotriacetic acid (NTA); sodium azide; or the likefurther is present with the oxidation color former. The spontaneouscolor development of the oxidation color former can be suppressed stillmore strongly by the presence of these substances. One kind of thesesubstances may be present with the oxidation color former, or two ormore kinds may be present with the oxidation color former.

[0013] Next, an oxidation color former reagent according to the presentinvention is a reagent solution including an aqueous solvent and anoxidation color former dissolved in the aqueous solvent, and at leastone of FAOD and POD further is dissolved in the aqueous solvent. Theoxidation color former may be, for example,N-(carboxymethylaminocarbonyl)-4,4′-bis (dimethylamino)diphenylaminesodium salt as in the above.

[0014] In such a reagent, spontaneous color development of the oxidationcolor former such as N-(carboxymethylaminocarbonyl)-4,4′-bis(dimethylamino)diphenylamine sodium salt is suppressed when the reagentis held or stored. Therefore, the necessity of preparing a reagent foreach use is eliminated so that various measurement reactions or the likeusingN-(carboxymethylaminocarbonyl)-4,4′-bis(dimethylamino)diphenylaminesodium salt, for example, can be carried out simply.

BRIEF DESCRIPTION OF DRAWINGS

[0015]FIG. 1 is a graph showing an absorbance spectrum of DA-64 whenFAOD is present with the DA-64 in a buffer in one example of astabilizing method according to the present invention.

[0016]FIG. 2 is a graph showing an absorbance spectrum of DA-64 when PODis present with the DA-64 in a buffer in another example of astabilizing method according to the present invention.

[0017]FIG. 3 is a graph showing an absorbance spectrum of DA-64 whenFAOD and POD are present with the DA-64 in a buffer in still anotherexample of a stabilizing method according to the present invention.

[0018]FIG. 4 is a graph showing an absorbance spectrum of DA-64 in abuffer in a comparative example.

BEST MODE FOR CARRYING OUT THE INVENTION

[0019] Hereinafter, the method of the present invention will bedescribed in detail with reference to the following examples, in whichN-(carboxymethyl aminocarbonyl)-4,4′-bis(dimethylamino)diphenylaminesodium salt is used as an oxidation color former.

[0020] The stabilization of N-(carboxymethylaminocarbonyl)-4,4′-bis(dimethylamino)diphenylamine sodium salt according to the presentinvention can be carried out, for example, by dissolvingN-(carboxymethylamino carbonyl)-4,4′-bis(dimethylamino)diphenylaminesodium salt and at least one of FAOD and POD in an aqueous solvent toprepare an aqueous solution of N-(carboxymethylaminocarbonyl)-4,4′-bis(dimethylamino)diphenylamine sodium salt.

[0021] As the FAOD, an enzyme catalyzing a reaction represented byFormula (1) below may be used, and the derivation thereof is notparticularly limited. For example, commercially available enzyme such asa product named FAOX-E (Kikkoman Corporation) and a product named FOD(Asahi Chemical Industry Co., Ltd.) can be used.

R¹—CO—CH₂—NH—R²+H₂O+O₂→R¹—CO—CHO+NH₂—R²+H₂O₂  (1)

[0022] In Formula (1), R¹ denotes a hydroxyl group or a residue derivedfrom the sugar before glycation (i.e., sugar residue). The sugar residue(R¹) is an aldose residue when the sugar before glycation is aldose, andis a ketose residue when the sugar before glycation is ketose. Forexample, when the sugar before glycation is glucose, it takes a fructosestructure after glycation by an Amadori rearrangement. In this case, thesugar residue (R¹) becomes a glucose residue (an aldose residue). Thissugar residue (R¹) can be represented, for example, by

[0023] —[CH(OH)]_(n)—CH₂OH

[0024] where n is an integer of 0 to 6.

[0025] In Formula (1), R² is not particularly limited. However, when thesubstrate is a glycated amino acid, a glycated peptide, or a glycatedprotein, for example, there is a difference between the case where anα-amino group is glycated and the case where an amino group other thanthe α-amino group is glycated.

[0026] In Formula (1), when an α-amino group is glycated, R² is an aminoacid residue or a peptide residue represented by Formula (2) below.

—CHR³—CO—R⁴  (2)

[0027] In Formula (2), R³ denotes an amino-acid side chain group. R⁴denotes a hydroxyl group, an amino acid residue, or a peptide residue,and can be represented, for example, by Formula (3) below. In Formula(3), n is an integer of 0 or more, and R³ denotes an amino-acid sidechain group as in the above.

−(NH—CHR³—CO)_(n)—OH  (3)

[0028] In Formula (1), when an amino group other than the α-amino groupis glycated (i.e., an amino-acid side chain group is glycated), R² canbe represented by Formula (4) below.

—R⁵—CH(NH—R⁶)—CO—R⁷  (4)

[0029] In Formula (4), R⁵ denotes a portion other than the glycatedamino group in the amino-acid side chain group. For example, when theglycated amino acid is lysine, R⁵ is as follows.

—CH₂—CH₂—CH₂—CH₂—

[0030] For another example, when the glycated amino acid is arginine, R⁵is as follows.

—CH₂—CH₂—CH₂—NH—CH(NH₂)—

[0031] In Formula (4), R⁶ denotes hydrogen, an amino acid residue, or apeptide residue, and can be represented, for example, by Formula (5)below. In Formula (5), n denotes an integer of 0 or more, and R³ denotesan amino-acid side chain group as in the above.

—(CO—CHR³—NH)_(n)—H  (5)

[0032] In Formula (4), R⁷ denotes a hydroxyl group, an amino acidresidue, or a peptide residue, and can be represented, for example, byFormula (6) below. In Formula (6), n is an integer of 0 or more, and R³denotes an amino-acid side chain group as in the above.

—(NH—CHR³—CO)_(n)—OH  (6)

[0033] As the POD, a known POD catalyzing a reaction represented byFormula (7) below can be used. In Formula (7), AH₂ denotes a substrateand A is not particularly limited.

AH₂+H₂O₂→A+2H₂O  (7)

[0034] The concentration of N-(carboxymethylaminocarbonyl)-4,4′-bis(dimethylamino)diphenylamine sodium salt in the aqueous solution is notparticularly limited, but is, for example, in the range from 1 to 10,000μmol/l, preferably 1 to 1000 μmol/l as described above, due to thesolubility ofN-(carboxymethylaminocarbonyl)-4,4′-bis(dimethylamino)diphenylaminesodium salt in water, etc.

[0035] When FAOD is added, its concentration is, for example, in therange from 0.002 to 200 g/l, preferably 0.01 to 50 g/l, and morepreferably 0.3 to 20 g/l, as described above. When indicated by enzymeactivity, it is, for example, in the range from 0.1 to 1000 KU/l,preferably 0.5 to 300 KU/l, and more preferably 1 to 150 KU/l.Furthermore, the amount of FAOD per 0.1 mmol ofN-(carboxymethylaminocarbonyl)-4,4′-bis(dimethylamino) diphenylaminesodium salt is, for example, in the range from 0.01 to 200 g, preferably0.1 to 50 g, and more preferably 0.6 to 20 g. When indicated by enzymeactivity, it is, for example, in the range from 0.5 to 1000 KU,preferably 1 to 300 KU, and more preferably 2 to 100 KU.

[0036] When POD is added, its concentration is, for example, in therange from 0.02 to 50 g/l, preferably 0.2 to 10 g/l, as described above.When indicated by enzyme activity, it is, for example, in the range from1 to 5000 KU/l, preferably 5 to 1000 KU/l. Furthermore, the amount ofPOD per 0.1 mmol ofN-(carboxymethylaminocarbonyl)-4,4′-bis(dimethylamino) diphenylaminesodium salt is, for example, in the range from 0.02 to 50 g, preferably0.2 to 10 g. When indicated by enzyme activity, it is, for example, inthe range from 1 to 5000 KU, preferably 5 to 1000 KU.

[0037] Alternatively, both FAOD and POD may be added. In this case, FAODand POD are added so that, for example, the FAOD concentration is in therange from 0.002 to 100 g/l while the POD concentration is in the rangefrom 0.02 to 50 g/l, preferably the FAOD concentration is in the rangefrom 0.01 to 50 g/l while the POD concentration is in the range from 0.2to 10 g/l, and more preferably the FAOD concentration is in the rangefrom 0.3 to 20 g/l while the POD concentration is in the range from 0.2to 10 g/l. When indicated by enzyme activity, they are added so that,for example, the FAOD concentration is in the range from 0.1 to 600 KU/lwhile the POD concentration is in the range from 1 to 5000 KU/l,preferably the FAOD concentration is in the range from 0.5 to 300 KU/lwhile the POD concentration is in the range from 5 to 1000 KU/l, andmore preferably the FAOD concentration is in the range from 1 to 150KU/l while the POD concentration is in the range from 5 to 1000 KU/l.

[0038] The amounts of FAOD and POD per 0.1 mmol of N-(carboxymethylaminocarbonyl)-4,4′-bis (dimethylamino)diphenylamine sodium salt are,for example, as follows: the amount of FAOD is in the range from 0.01 to100 g while the amount of POD is in the range from 0.02 to 50 g,preferably the amount of FAOD is in the range from 0.1 to 50 g while theamount of POD is in the range from 0.2 to 10 g, and more preferably theamount of FAOD is in the range from 0.6 to 20 g while the amount of PODis in the range from 0.2 to 10 g. When indicated by enzyme activity,they are as follows, for example: the amount of FAOD is in the rangefrom 0.5 to 600 KU while the amount of POD is in the range from 1 to5000 KU, preferably the amount of FAOD is in the range from 1 to 300 KUwhile the amount of POD is in the range from 5 to 1000 KU, and morepreferably the amount of FAOD is in the range from 2 to 100 KU while theamount of POD is in the range from 5 to 1000 KU.

[0039] Furthermore, FAOD (A) and POD (B) are added so that they arepresent at a weight ratio (A:B), for example, in the range from 100:0 to0:100.

[0040] As the aqueous solvent, water, various buffers, and the like canbe used, for example. Among these, various buffers are preferablebecause spontaneous color development ofN-(carboxymethylaminocarbonyl)-4,4′-bis(dimethylamino)diphenylaminesodium salt can be suppressed more strongly. Examples of the bufferinclude ADA buffer, Tris-HCl buffer, Bis-Tris buffer, glycylglycinebuffer, Bicine buffer, and a phosphate buffer such as potassiumphosphate buffer (KPB) as described above, and HEPES buffer, HEPSObuffer, and the like. Among these, ADA buffer, Tris-HCl buffer, andphosphate buffer are preferable. The pH of the buffer is, for example,in the range from 5.0 to 9.0, preferably 6.0 to 8.0.

[0041] The concentration of the buffer is not particularly limited, andis, for example, in the range from 1 to 1000 mmol/l. However, preferablyit is in the range from 50 to 800 mmol/l, more preferably 100 to 500mmol/l, because spontaneous color development ofN-(carboxymethylaminocarbonyl)-4,4′-bis(dimethylamino)diphenylaminesodium salt can be suppressed more strongly when the concentration ofthe buffer is relatively high.

[0042] Furthermore, the pH of the aqueous solution prepared is notparticularly limited, but is, for example, in the range from 5.0 to 9.0,preferably 6.0 to 8.0.

[0043] Such an aqueous solution ofN-(carboxymethylaminocarbonyl)-4,4′-bis(dimethylamino)diphenylaminesodium salt can be stabilized when at least one of FAOD and POD ispresent with N-(carboxymethylaminocarbonyl)-4,4′-bis(dimethylamino)diphenylamine sodium salt. Thus, it becomespossible to store N-(carboxymethylaminocarbonyl)-4,4′-bis(dimethylamino)diphenylamine sodium salt as a solution. The storage temperature is notparticularly limited, but is, for example, in the range from 0° C. to40° C., preferably 0° C. to 25° C., and more preferably 0° C. to 10° C.

[0044] When the aqueous solution is stored at 10° C. without addingeither FAOD or POD, the absorbance measured at 727 nm as the absorptionwavelength of N-(carboxymethylaminocarbonyl)-4,4′-bis(dimethylamino)diphenylamine sodium salt that has developed color increases, forexample, by a factor of 2 to 3 after the storage for 5 days and by afactor of 10 to 11 after the storage for 18 days. In contrast, when theaqueous solution stabilized by the method of the present invention isstored at 10° C., the spontaneous color development can be suppressed,for example, for 9 days, preferably for 1 to 5 days.

[0045] Furthermore, in the method of the present invention, not onlyFAOD and POD, but also an antioxidant, a chelating agent, sodium azide,and the like as described above may be present with the oxidation colorformer. Among these, a chelating agent and sodium azide are preferable.

[0046] Examples of the antioxidant include VE, potassium erythorbate,and potassium sorbate as described above. The antioxidant is added sothat its concentration falls in the range from, for example, 0.1 to 1000μmol/l, preferably 0.2 to 100 μmol/l, and more preferably 0.5 to 20Ξmol/l. Furthermore, the amount of the antioxidant is in the range from,for example, 0.1 to 100 μmol, preferably 0.2 to 20 μmol per 0.1 mmol/lof N-(carboxymethyl aminocarbonyl)-4,4′-bis(dimethylamino)diphenylaminesodium salt.

[0047] Examples of the chelating agent include EDTA, DTPA, CyDTA GEDTA,and NTA as described above. The chelating agent is added so that itsconcentration falls in the range from, for example, 0.01 to 20 mmol/l,preferably 0.05 to 10 mmol/l, and more preferably 0.1 to 5 mmol/l.Furthermore, the amount of the chelating agent is in the range from, forexample, 0.02 to 15 mmol, preferably 0.05 to 10 mmol, and morepreferably 0.1 to 5 mmol per 0.1 mmol ofN-(carboxymethylaminocarbonyl)-4,4′-bis(dimethylamino)diphenylaminesodium salt.

[0048] Sodium azide is added so that its concentration falls in therange from, for example, 0.01 to 20 mmol/l, preferably 0.05 to 10mmol/l, and more preferably 0.1 to 5 mmol/l. Furthermore, the amount ofsodium azide is in the range from, for example, 0.01 to 10 mmol,preferably 0.05 to 5 mmol, and more preferably 0.1 to 2 mmol per 0.1mmol of N-(carboxymethylamino carbonyl)-4,4′-bis(dimethylamino)diphenylamine sodium salt.

[0049] N-(carboxymethylaminocarbonyl)-4,4′-bis(dimethylamino)diphenylamine sodium salt stabilized in the above-described manner isuseful as a liquid reagent because the spontaneous color development issuppressed as described above even if it is stored as a solution for along time. The application of theN-(carboxymethylaminocarbonyl)-4,4′-bis(dimethylamino) diphenylaminesodium salt reagent is not particularly limited. For example, thereagent may be used as a color-developing substrate in a redox reactionor the like as described above. When the reagent is used in such areaction, an increase in background absorbance in the measurement of anabsorbance is suppressed because the spontaneous color development issuppressed as described above. Therefore, the reagent can improve theaccuracy of various measurements. Moreover, as in the stabilizing methodof the present invention, it also is preferable to add various buffers,an antioxidant, a chelating agent, sodium azide, and the like to thereagent.

EXAMPLES Examples 1 to 3 and Comparative Example 1

[0050] In Examples 1 to 3, N-(carboxymethylaminocarbonyl)-4,4′-bis(dimethylamino)diphenylamine sodium salt was present with at least oneof FAOD and POD in Tris-HCl buffer, and the stability (change inabsorbance) of the solution was examined.

[0051] First, N-(carboxymethylaminocarbonyl)-4,4′-bis(dimethylamino)diphenylamine sodium salt (product name DA-64, manufactured by Wako PureChemical Industries, Ltd., hereinafter referred to as “DA-64”) was addedto 200 mM Tris-HCl buffer (pH 7.0) so that its concentration became0.088 mM. Then, by adding FAOD (product name FOD, manufactured by AsahiChemical Industry Co., Ltd.) and/or POD (manufactured by Toyobo Co.,Ltd.) to the thus-obtained DA-64 solution, samples containing the FAODand/or the POD at the following concentrations were prepared. Theabsorption spectrum was measured with a biochemical automatic analysisapparatus (product name JCA-BM 8, manufactured by Japan Electron OpticsLaboratory Co. Ltd.) after storing the samples at 10° C. forpredetermined periods (immediately after the preparation, 5 days, 9days, and 18 days). On the other hand, in Comparative Example 1, asample was prepared in the same manner as in Examples 1 to 3 except thatneither FAOD nor POD was added, and the absorption spectrum was measuredalso in the same manner. FAOD Concentration POD Concentration Example 10.09 g/l (22 KU/l) — Example 2 — 0.37 g/l (15 KU/l) Example 3 0.09 g/l(22 KU/l) 0.37 g/l (15 KU/l) Comparative Example 1 — —

[0052] The results are shown in FIGS. 1 to 4. FIGS. 1 to 4 are graphsshowing the absorption spectrum measured after the storage for eachpredetermined period, wherein FIG. 1 shows the data of Example 1, FIG. 2shows the data of Example 2, FIG. 3 shows the data of Example 3, andFIG. 4 shows the data of Comparative Example 1.

[0053] As can be seem from the drawings, in Examples 1 to 3 where atleast one of the FAOD and POD was added, an increase in absorbancemeasured at 727 nm as the absorption wavelength of DA-64 that haddeveloped color was suppressed to about ½ to {fraction (1/10)} of thatin Comparative Example 1. In particular, in Example 3 where both theFAOD and POD were added, the spontaneous color development wassuppressed most significantly.

INDUSTRIAL APPLICABILITY

[0054] As specifically described above, according to a method of thepresent invention, an oxidation color former such asN-(carboxymethylamino carbonyl)-4,4′-bis(dimethylamino)diphenylaminesodium salt can be stored stably as a solution. Therefore, when a liquidreagent of an oxidation color former such asN-(carboxymethylaminocarbonyl)-4,4′-bis(dimethylamino) diphenylaminesodium salt is needed, it is not necessary to prepare a reagent for eachuse. This lowers the cost of the reagent and also simplifies theoperation.

1. A method of stabilizing an oxidation color former in a solution,comprising: making at least one of a fructosyl amino acid oxidase and aperoxidase present with the oxidation color former in the solution. 2.The method according to claim 1, wherein the oxidation color former isN-(carboxymethylaminocarbonyl)-4,4′-bis(dimethylamino)diphenylaminesodium salt.
 3. The method according to claim 1, wherein a concentrationof the oxidation color former is in a range from 1 to 10,000 μmol/l. 4.The method according to claim 1, wherein a concentration of thefructosyl amino acid oxidase is in a range from 0.002 to 200 g/l or 0.1to 1000 KU/l.
 5. The method according to claim 1, wherein aconcentration of the peroxidase is in a range from 0.02 to 50 g/l or 1to 5000 KU/l.
 6. The method according to claim 1, wherein an amount ofthe fructosyl amino acid oxidase per 0.1 mmol of the oxidation colorformer is in a range from 0.01 to 200 g or 0.5 to 1000 KU, and an amountof the peroxidase per 0.1 mmol of the oxidation color former is in arange from 0.02 to 50 g or 1 to 5000 KU.
 7. The method according toclaim 1, wherein the solution contains at least one buffer selected fromthe group consisting of ADA buffer, Tris-HCl buffer, Bis-Tris buffer,glycylglycine buffer, Bicine buffer, and phosphate buffer.
 8. The methodaccording to claim 1, further comprising: making at least oneantioxidant selected from the group consisting of α-tocopherol acetate(VE), potassium erythorbate, and potassium sorbate present with theoxidation color former.
 9. The method according to claim 1, furthercomprising: making at least one chelating agent selected from the groupconsisting of ethylenediaminetetraacetic acid (EDTA),diethylenetriaminepentaacetic acid (DTPA),trans-1,2-diaminocyclohexane-N,N,N′,N′-tetraacetic acid (CyDTA),O,O′-bis(2-aminoethyl)ethyleneglycol-N,N,N′,N′-tetraacetic acid (GEDTA),and nitrilotriacetic acid (NTA) present with the oxidation color former.10. The method according to claim 1, further comprising: making sodiumazide present with the oxidation color former.
 11. The method accordingto claim 1, wherein a pH of the solution is in a range from 5.0 to 9.0.12. The method according to claim 1, wherein a temperature of thesolution is in a range from 0° C. to 40° C.
 13. A reagent solutioncomprising an aqueous solvent and an oxidation color former dissolved inthe aqueous solvent, wherein at least one of a fructosyl amino acidoxidase and a peroxidase further is dissolved in the aqueous solvent.14. The reagent solution according to claim 13, wherein the oxidationcolor former is N-(carboxymethylaminocarbonyl)-4,4′-bis(dimethylamino)diphenylamine sodium salt.